Project Summary The control of mRNA stability is a critical determinant in the post-transcriptional regulation of eukaryotic gene expression. Even minor alterations in mRNA stability can have profound consequences and may manifest as clinical phenotypes as illustrated by the ability of aberrantly expressed proto-oncogenes that can give rise to malignancies. Eukaryotic mRNAs are generally thought to possess an N7 methyl guanosine (m7G) cap at their 5 end to promote their stability and translation. However, our recent demonstration that mammalian mRNAs can also carry a 5-end nicotinamide adenine dinucleotide (NAD) cap that in contrast to the m7G cap promotes mRNA decay, provides a new paradigm for mRNA 5 end processing and the contribution of nucleotide metabolites in mRNA turnover. We now demonstrate that the redox state of NAD can also modulate 3 RNA decay with free NAD functioning as a cofactor to enhance RNA decay and potentially providing a link to cellular energetics. Moreover, flavin adenine diphosphate (FAD) can also serve as a 5 cap on mammalian RNAs with Nudt16 and DXO hydrolases functioning as proteins that can remove the FAD cap (deFADding) in vitro. We will build on these novel findings throughout this proposal within three specific aims. The first will address the functional role of free NAD on the control of 3 end RNA decay in vitro and delineate the molecular mechanism involved in its stimulation of decay. The second will deduce changes in mRNA decay as a consequence of altered NAD levels in cells and assess the regulatory role imparted by stress conditions in modulating RNA decay through the control of NAD levels. In the last aim, we establish FAD cap as an alternative RNA cap, identify FAD-capped RNAs and decipher the role of FAD caps and the deFADding enzymes in cells. Collectively, the proposed studies will provide insight into a heretofore unknown fundamental post-transcriptional regulatory mechanism and will provide the framework for potential novel avenues to control gene expression in normal and disease states.